In the initial stage of host cell carbohydrate metabolism, that is, glycolysis, each glucose molecule is converted to two molecules of pyruvate in the cytosol. The chemical reactions that convert glucose to pyruvate are referred to as the Embden-Meyerhoff pathway. All of the metabolic intermediates between the initial carbohydrate and the final product, pyruvate, are phosphorylated compounds. The final stage of oxidation of carbohydrates, the citric acid cycle, is a complex set of reactions that also takes place in the cytosol. The reactions in the Embden-Meyerhoff pathway and citric acid cycle result in the conversion of carbohydrate molecules to CO2 molecules with the concomitant reduction of NAD+ to NADH molecules and the formation of ATP.
The central metabolic routes produce NADH or NADPH. In general NADPH is utilized in biosynthetic reactions and NADH is rapidly reoxidized in two ways:    (1) In fermentative pathways by the direct reduction of organic metabolites.    (2) In respiratory processes by electron transport through a respiratory chain to a terminal electron acceptor. This acceptor is usually O2, but in some cases can be productive ions, including nitrate and sulfate. In all respiratory processes, ATP is generated.Some bacteria posses the ability to oxidize some substrates extracellularly, producing useful oxidation products such as L-sorbose, D-gluconate, keto-gluconates, etc. Such oxidation reactions are called productive fermentation since they involve incomplete substrate oxidation, accompanying accumulation of corresponding oxidation product in large amounts in the growth medium. The oxidation reaction is coupled to the respiratory chain of the microorganism. (Bacterial Metabolism 2nd Edition (1985) Springer-Verlag, New York, N.Y.).
Bacteria which ferment glucose through the Embden-Meyerhof pathway, such as members of Enterobacteriacea and Vibrionaceae, are described in Bouvet, et al. (1989) International Journal of Systematic Bacteriology, 39:61-67. Pathways for metabolism of ketoaldonic acids in Erwinia sp. are described in Truesdell, et al, (1991) Journal of Bacteriology, 173:6651-6656.
Host cells having mutations in enzymes involved in glycolysis have been described. Yeast having mutations in glucokinase are described in Harrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379-383; Wedlock, et al., (1989) J. Gen. Microbiol. 135: 2013-2018; and Walsh et al. (1983) J. Bacteriol. 154:1002-1004. Bacteria deficient in glucokinase have been described. Pediococcus sp. deficient in glucokinase is described in Japanese patent publication JP 4267860. Bacillus sphaericus lacking glucokinase is described in Russell et al. (1989) Appl. Environ. Microbiol. 55: 294-297. Penicillium chrysogenum deficient in glucokinase is described in Barredo et al. (1988) Antimicrob. Agents-Chemother 32:1061-1067. A glucokinase-deficient mutant of Zymomonas mobilis is described in DiMarco et al. (1985) Appl. Environ. Microbiol. 49:151-157.
Many bacteria posses an active transport system known as Phosphotransferase transport System (PTS) that couples the transport of a carbon source to its phosphorylation. In this system, the phosphoryl group is transferred sequentially from phosphoenolpyruvate (PEP) to enzyme I and from enzyme I to protein HPr. The actual translocation step is catalyzed by a family of membrane bound enzymes (called enzyme II), each of which is specific for one or a few carbon sources. Considering that PTS consumes PEP to phosphorylate the carbon source, and PEP is a central metabolite used in for many biosynthetic reactions, it may decrease the efficiency of conversion of a carbon source into a desired product. this transport system has been replaced by a permease and glucokinase from an heterologous origin as described by Parker et al. (1995) Mol. Microbiol. 15: 795-802. or homologous origin as reported by Flores et al. (1996) Nat. Biotechnol. 14: 620-623. In both of these 2 examples, the function of the PTS system for glucose transport and phosphorylation was replaced by a glucose permease and a glucokinase activities.
Products of commercial interest that have been produced biocatalytically in genetically engineered host cells include intermediates of L-ascorbic acid; 1,3-propanediol; glycerol; D-gluconic acid; aromatic amino acids; 3-deozy-D-arabino-heptulosonate 7-phosphate (DAHP); and catechol, among others.
L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical and food industry as a vitamin and antioxidant. The synthesis of ASA has received considerable attention over many years due to its relatively large market volume and high value as a specialty chemical.
The Reichstein-Grussner method, a chemical synthesis route from glucose to ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328). Lazarus et al. (1989, “Vitamin C: Bioconversion via a Recombinant DNA Approach”, Genetics and Molecular Biology of Industrial Microorganisms, American Society for Microbiology, Washington D.C. Edited by C. L. Hershberger) disclose a bioconversion method for production of an intermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically converted to ASA. This bioconversion of carbon source to KLG involves a variety of intermediates, the enzymatic process being associated with co-factor dependent 2,5-DKG reductase activity (2,5-DKGR or DKGR).
Many bacterial species have been found to contain DKGR, particularly members of the Coryneform group, including the genera Corynebacterium, Brevibacterium, and Arthrobacter. DKGR obtained from Corynebacterium sp. strain SHS752001 is described in Grindley et al. (1988, Applied and Environmental Microbiology 54: 1770-1775). DKGR from Erwinia herbicola is disclosed in U.S. Pat. No. 5,008,193 to Anderson et al. Other reductases are disclosed in U.S. Pat. Nos. 5,795,761; 5,376,544; 5,583,025; 4,757,012; 4,758,514; 5,004,690; and 5,032,514.
1,3-Propanediol is an intermediate in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. The production of 1,3-propanediol is described in U.S. Pat. Nos. 6,025,184 and 5,686,286. 1,3-propanediol can be produced by the fermentation of glycerol. The production of glycerol is described in WO 99/28480 and WO 98/21340.
D-gluconic acid and its derivatives have been used commercially as agents in textile bleaching and detergents. The production of D-gluconic acid in Bacillus species lacking gluconokinase activity and having high glucose dehydrogenase activity is described in WO 92/18637.
The production of members of the aspartate family of amino acids is described in U.S. Pat. No. 5,939,307. The production of riboflavin (Vitamin B2) is described in WO 99/61623.
Many cyclic and aromatic metabolites are derived from DHAP including tyrosine, tryptophan and phenylalanine. The production of DAHP is described in U.S. Pat. No. 5,985,617. Catechol is a starting material for the synthesis of pharmaceuticals, pesticides, flavors, fragrances and polymerization inhibitors. The production of catechol is described in U.S. Pat. No. 5,272,073.
However, there are still problems associated with these production methodologies. One such problem is the diversion of carbon substrates from the desired productive pathways to the catabolic pathways. Such diversion results in the loss of available carbon substrate material for conversion to the desired productive pathway products and resultant energy costs, ATP or NADPH, associated with the transport or phosphorylation of the substrate for catabolic pathway use.
In spite of the advances made in the production of products by host cells, there remains a need for improved host cells for use in the production of desired products. The present invention addresses that need.